G protein-coupled receptors (GPCRs) represent the largest family of signal-transducing molecules known. For example, GPCRs comprise more than 4% of the genes in Caenorhabditis elegans. GPCRs convey signals for light and many extracellular regulatory molecules, such as, hormones, growth factors and neurotransmitters, that regulate every cell in the body. Dysregulation of GPCRs has been found in a growing number of human diseases and GPCRs have been estimated to be the targets of more than 30% of the drugs used in clinical medicine today. Thus, understanding how GPCRs function at the molecular level is an important goal of biological research. We have used receptors for thyrotropin-releasing hormone (TRH) (TRH-Rs) as model GPCRs to study their structure and function. During this year, we have studied several new aspects of TRH-R structure and function. We compared several aspects of the biology of the two mouse TRH receptor types 1 (mTRH-R1) and 2 (mTRH-R2), which are 50% identical at the amino acid level. mTRH-R2 shows a higher basal signaling activity than mTRH-R1. We used chimeras/mutants of these receptors to gain insight into the properties of the receptors that influence internalization and basal signaling. We observed that receptors with more rapid internalization rates showed relatively higher basal signaling activities whereas receptors with lower basal signaling activities showed slower internalization rates. These data suggest that similar receptor conformations are required for efficient coupling to signaling G proteins and to proteins involved in internalization. In another aspect of the project, we synthesized several novel TRH analogs in which the N-terminal polyglutamic acid residue was replaced with polycarboxylic acids and the central histidine was modified with substituted imidazole derivatives and studied their biological properties. These analogs provided new insights into the size of the binding pocket for TRH within TRH-R1. Lastly, we have been exploring differences in the binding of mTRH-R1 and mTRH-R2 for competitive, small organic molecule, inverse agonists; there are no discernible differences between the two receptors for binding peptidic agonists. We have tentatively identified a new series of benzodiazepine drugs that discriminate between mTRH-R1 and mTRH-R2.